Allium sativum L. (garlic) has received a growing interest as both a food plant and folk remedy with promising antitumor and chemopreventive potential. So far, a large body of research has been conducted on garlic, mostly focusing on the bulbs, yet its roots remain unstudied and are discarded in considerable amounts as vegetable wastes. Accordingly, for the first time, this study examined the potential of A. sativum roots and their green synthesized silver nanoparticles (AgNPs) against hepatocellular carcinoma (HepG-2), breast cancer (Mcf-7), and colon cancer (Caco-2) cells lines. The formation of AgNPs was confirmed using a Transmission Electron Microscope (TEM), UV‒Visible spectroscopy, and Fourier-Transform Infrared Spectroscopy (FT-IR). Overall, the total ethanolic extract of A. sativum roots (TEEASR) significantly inhibited the growth of Caco-2, Mcf-7, and HepG-2 cells with IC₅₀ values of 2.11 ± 0.03, 21.47 ± 0.15, and 45.12 ± 0.76 µg/mL, respectively, whereas AgNPs exhibited more prominent cytotoxic activity, which in the case of Caco-2 cells, was noticeably greater than doxorubicin (IC₅₀= 0.47 ± 0.04 vs. 2.12 ± 0.04 µg/mL). Additionally, HPLC‒HESI‒HRMS analysis of TEEASR revealed the presence of a variety of metabolites with reported anticancer effects, largely dominated by organosulfur species. Interestingly, the characterized compounds showed moderate to promising binding interactions with the active site of human inositol phosphate multikinase (HIPMK), especially γ-glutamylphenylalanine (6) and allixin (7); highlighting their potential contribution to the notable cytotoxic properties of TEEASR as HIPMK inhibitors. These results might pave the way for the possible therapeutic and pharmaceutical applications of garlic by-products, including the roots, and their biogenic nanoparticles.